The Hypothetical Protein ‘All4779’, and Not the Annotated ‘Alr0088’ and ‘Alr7579’ Proteins, Is the Major Typical Single-Stranded DNA Binding Protein of the Cyanobacterium, Anabaena sp. PCC7120

Anurag Kirti,Hema Rajaram,Shree Kumar Apte,Sergey Korolev

doi:10.1371/journal.pone.0093592

Anurag Kirti, Hema Rajaram + Show 2 more

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https://doi.org/10.1371/journal.pone.0093592

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Journal: PLoS ONE	Publication Date: Apr 4, 2014
Citations: 30	License type: CC BY 4.0

Affiliation: Bhabha Atomic Research Centre

Abstract

Single-stranded DNA binding (SSB) proteins are essential for all DNA-dependent cellular processes. Typical SSB proteins have an N-terminal Oligonucleotide-Binding (OB) fold, a Proline/Glycine rich region, followed by a C-terminal acidic tail. In the genome of the heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120, alr0088 and alr7579 are annotated as coding for SSB, but are truncated and have only the OB-fold. In silico analysis of whole genome of Anabaena sp. strain PCC7120 revealed the presence of another ORF ‘all4779’, annotated as a hypothetical protein, but having an N-terminal OB-fold, a P/G-rich region and a C-terminal acidic tail. Biochemical characterisation of all three purified recombinant proteins revealed that they exist either as monomer or dimer and bind ssDNA, but differently. The All4779 bound ssDNA in two binding modes i.e. (All4779)35 and (All4779)66 depending on salt concentration and with a binding affinity similar to that of Escherichia coli SSB. On the other hand, Alr0088 bound in a single binding mode of 50-mer and Alr7579 only to large stretches of ssDNA, suggesting that All4779, in all likelihood, is the major typical bacterial SSB in Anabaena. Overexpression of All4779 in Anabaena sp. strain PCC7120 led to enhancement of tolerance to DNA-damaging stresses, such as γ-rays, UV-irradiation, desiccation and mitomycinC exposure. The tolerance appears to be a consequence of reduced DNA damage or efficient DNA repair due to increased availability of All4779. The ORF all4779 is proposed to be re-annotated as Anabaena ssb gene.

Highlights

Single-stranded DNA-binding proteins (SSB) are ubiquitous proteins found in all bacteria
Among the naturally nontransformable bacteria, the smallest known bacterial SSB are from the thermophilic bacteria, Thermotoga maritima (TmaSSB) and T. neapolitana (TneSSB) consisting of 141 and 142 amino acids respectively, having a single OB-fold domain and a C-terminal domain with the conserved DEPPF terminal amino acids [21]
All4779 had a long Cterminal region similar to that observed for EcoSSB which comprised of a proline-rich region (19 residues) with two glycine residues, as compared to EcoSSB which is glycine rich (21 residues) and has 8 prolines in the corresponding region (Figure 1B)

Summary

Introduction

Single-stranded DNA-binding proteins (SSB) are ubiquitous proteins found in all bacteria. Generation of Plasmid Constructs for Overexpression of Proteins in E. coli Different amplicons (alr0088, alr7579, all4779) were generated by PCR amplification of Anabaena 7120 genomic DNA (100 ng) using gene specific primers (as indicated in Table 1), 1 mM dNTPs and 1U Taq DNA polymerase in Taq buffer (Bangalore Genei, India).

Results

Conclusion